The objective of this research is to broaden understanding of the glutamate uptake system in the synaptic vesicle, particularly to investigate its regulation. Glutamate is now recognized as the major excitatory neurotransmitter in the central nervous system. As such, proper glutamate synaptic transmission is required not only for basic neuronal communication, but also for learning and memory formation. Moreover, a variety of evidence indicates that aberrant glutamate transmission is involved in many types of pathophysiologies in the central nervous system. The vesicular glutamate uptake system is highly specific for glutamate and considered to play a critical role in directing glutamate to the neurotransmitter pathway away from the metabolic pathway. Hence, it has been postulated to be subject to regulation. The applicant's laboratory previously provided evidence for an endogenous proteinaceous factor that inhibits vesicular glutamate uptake. This factor has now been purified to apparent homogeneity, revealing that it is indeed a potent protein inhibitor apparently specific for glutamate uptake into synaptic vesicles. Sequence analyses suggest that this inhibitory protein, referred to as glustoreducin (GSR), is derived from alpha-fodrin, a major cytoskeletal protein component. The PI proposes to (1) demonstrate the relationship between the production of GSR and the acquisition of the ability to regulate vesicular glutamate accumulation, by correlating, under various incubation conditions, the GSR production with the reduction of [3/H]glutamate accumulated into synaptic vesicles; (2) further characterize GSR with respect to (a) the inhibition constant by kinetic experiments; (b) neurotransmitter specificity by examining the effect of GSR on vesicular uptake of GABA and glycine; and (c) site of action by examining the effect on GSR on H+ pump ATPase activity, membrane potential, and glutamate efflux; and (3) demonstrate that GSR is generated from alpha-fodrin under physiologically relevant conditions, by incubating synaptosomes under various conditions, followed by gel blot radioimmunoassay. This research is expected to contribute to a better understanding of presynaptic regulation of glutamate transmission. It is also hoped that this investigation will ultimately provide new insights into some of the neurological and psychiatric disorders involving abnormal glutamate transmission.